MHC-II genes are expressed in a tissue-specific manner and are regulated both developmentally and by the actions of immune cytokines. Proper regulation of the MHC-II genes is central to a healthy and active immune system as MHC-II proteins present processed peptide antigens to helper T cells, resulting in the selection of T cells, activation of T cells, and initiation of T cell regulatory and humoral responses. The goal of this program is to elucidate the molecular mechanisms that regulate this system. During the previous funding period, we found that the MHC-II region is divided by a series of transcriptional insulators, sequences that organize genes and chromatin into regulatory domains. These insulators interact with each other, forming a series of foci and basic architecture of the locus. But unlike most other insulators, these MHCII insulators interact directly with active MHC-II promoters that have the MHC-II master regulator CIITA bound. MHC-II gene transcription and these interactions are dependent on the insulator-binding factor CTCF. Our observations establish a new paradigm for how these genes are regulated. Importantly, we do not know critical aspects of how MHCII- insulators function. For example, are the MHCII-insulators interchangeable or are they specific for a certain MHC-II genes? Is position/location of the insulator important? How can CTCF interact with itself and with CIITA; does it use separate domains for these interactions? We have found that the chromosomal segregation complex cohesin is associated with MHCII insulators, and that depletion of cohesin subunits reduces HLA-DRA expression, but we do not know how cohesin functions at these sites. Aims in this proposal will answer these questions and define the basic parameters of these elements. Additionally, we will extend our investigation and ask do other MHC-II loci, such as the Ia region of the mouse, use the same paradigm to regulate its MHC- II genes; and if so, does a loss of an MHCII-insulator in the mouse alter the in vivo expression pattern of MHC- II genes and the ability to respond to antigen? We will answer this question by characterizing the mouse MHC- II locus and create a mouse that has an MHCII insulator deleted from its genome. Because MHC-II genes are highly regulated and their expression is required to initiate and regulate immunity, the goal of this application is to define the mechanisms by which these novel MHCII-insulator elements function. Ultimately, the mechanisms and interactions defined here can be used as potential targets for the design of treatments that specifically modulate MHC-II-based immune responsiveness for infectious disease, autoimmune disease, cancer, and organ transplantation.